The purpose of this project is to identify genes of the diarrheagenic bacteria enteropathogenic E. coli (EPEC) by testing specific knockout mutants for virulence in human volunteers. In situ expression of putative virulence genes will also be tested by analyzing mRNA from endoscopically-directed biopsies of the jejunum from EPEC-infected volunteers. In particular, this study will focus on the bundle-forming pili (BFP), an organelle that we hypothesize is necessary for efficient intestinal colonization. Knockout mutations of the BFP gene cluster have been prepared by allelic replacement and these will be compared for virulence, in human volunteers, with the parent wild-type strain. If BFP is confirmed to be a virulence determinant, vaccine candidates containing the BFP antigen will be prepared and tested in humans.